Method for producing hexanor-5,9-seco-pregnan-5-oic acid

ABSTRACT

The present invention relates to a microbiological method for producing 20beta-hydroxy-9-oxo-1,2,3,4,10,19-hexanor-5,9-secopregnan-5-oic acid, an intermediate for known steroids, from 3beta, 20beta-dihydroxypregn-5-ene or 2beta, 20-dihydroxypregn-5ene.

United States Patent [1 1 Bec her et a1.

METHOD FOR PRODUCING HEXANOR-5,9-SECO-PREGNAN-5-OIC ACID Inventors:Elisabeth Becher, Basel; Arno Johannes Schocher, Benken; Erich Widmer,Arlesheim, all of Appl. 190.; 389,041

Foreign Application Priority Data- Aug. 31, 1972 Switzerland 1. 12838/72U.S. Cl. 195/51 R, 260/51-4 R Int. Cl C12d 1/02 Field of Search 195/51R, 51 G [451 Dec. 10, 1974 [56] References Cited UNITED STATES PATENTS3,379,621 4/1968 Casas-Campillo 195/51 G 3,616,228 -10/1971 Schuberteta] 195/51 R Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, orFirmSamuel L. Welt: Jon S.

Saxe; W. H. Epstein [57 ABSTRACT The present invention relates to amicrobiological method for producing 2Obeta-hydr0xy- 9-ox0-1,2,3,4,10,l9-hexanor-5,9-seco-pregnan-5-oic acid, an

intermediate for known steroids, from 3bet'a,ZObeta-dihydroXypregn-S-ene or 2beta, ZO-dihydroxypregn-S-ene.

4 Claims, N0 Drawings METHOD FOR PRODUCING HEXANOR-S,9-SECO-PREGNANB-DTE E C I1) SUMMARY OF THE INVENTION In accordance with thisinvention, a process is provided for the preparation of20beta-hydroxy-9-oxo- The process of this invention is carried out byfermenting 3beta,ZObeta-dihydroxypregn-S-ene or20betahydroxy-pregn-4-en-3-one with Mycobacterium phlei ATCC 19249.

The compound of formula I is utilized as an intermediate in thepreparation of known steroids by procedures described in Belgian Pat.No. 741,826.

DETAILED DESCRIPTION OF THE INVENTION In the pictorial representation ofthe compound of formula I, the symbol indicates a substituent which isin the alpha-orientation, i.e., below the plane ofthe molecule and thesymbol (B) indicates a substituent which is above the plane of themolecule.

By the term Mycobacterium phlei ATCC 19249 it is intended to cover allmicroorganisms of the genus Mycobacterium phlei which produce thecompound of formula land which cannot be definitely differentiated fromthe strain deposited with the American Type Culture Collection,Rockville, Maryland under ATCC No. 19249 and its subculturesincludingmutants and variants. According to the process provided by thepresent invention, the hexanor-S,9-seco-pregnan-5-oic acid of formula Iis manufactured by fermenting 3beta,20betadihydrOXypregn-S-ene or2Obeta-hydroxypregn-4-en- 3-one with Mycobacterium phlei ATCC 19249adapted to aliphatic hydrocarbons.

The adaption of Mycobacterium phlei ATCC 19249 to aliphatic hydrocarbonscan be carried out in a manas the carbon source, thehexanor-S,9-seco-pregnan-5- oic acid of formula I. Those cultures whichshow no growth on such media are used for the present fermentation.

The present process can be carried out by any conventional procedure ofaerobic fermentation. Especially suitable culture substrates are liquidmedia which contain a source of assimilable nitrogen and a source ofassimilable carbon as well as inorganic salts. Any conventional sourceof assimilable nitrogen and assimilablecarbon can be utilized. As thesource of assimilable nitrogen there can be used animal, vegetable,microbial and inorganic nitrogen compounds such as meat extracts,peptone, cornsteep, yeast extract, glycine and sodium nitrate. As thesource of assimilable carbon there can be used sugars such as glucoseand, especially, the steroid starting material to be fermented. Thenutrient medium may, if desired, also contain trace elements such asiron, sulfur and phosphorus as well as growth factors (e.g., vitaminssuch as biotin or pyridoxine) or auxins (e.g., indolylacetic acid). Inorder to protect against infections, the medium can be sterilized and/orcan be provided with substances such as benzoates, antibiotics, etc.,which inhibit the growth of foreign organisms. The fermentation ispreferably carried out in a neutral or weakly acidic pI-I-range (e.g.,

at pH 6-7). The fermentation can be carried out at a temperature ofbetween 18C. and 40C. It is preferably carried out at about 28C.

The steroid used as the starting material is preferably added to thefermentation culture in solution. Any conventional inert organic solventcan be used as the solvent medium in accordance with this invention.Among the preferred solvents are included dimethyl sulfoxide,dimethylformamide, ethanol or acetone. It has proved to be advantageousto add the steroid gradually to the fermentation solution and to waitfor the complete decomposition of the previously added steroid beforeeach new addition.

At. the end of the fermentation, the hexanor-5,9- seco-pregnan-S-oicacid aforesaid can be isolated from the culture solution by conventionalprocedures.

ner known per se by inoculating the microorganism from culturesubstrates which contain glucose on to those substrates in which theglucose is replaced in increasing quantity by aliphatic hydrocarbons,especially C -C alkanes.

The term mutants as used herein can be spontaneous' mutants or mutantsproduced in a physical or chemical manner. Mutants can be produced byirradiation (e.g., with ultraviolet light or gamma rays) or by treatmentwith mutating agents (e.g., N-methyl-nitronitrosoguanidine orbromouracil). Spontaneous mutants are preferably used in the presentprocess. The selection of the mutants can be carried out by spreadingsingle colonies on agar plates which contain, as the carbon source, thesteroid starting material to be fermented. Single colonies obtained fromsuch agar substrates are finally spread on agar plates which contain,

EXAMPLE 1 Production of the fermentation culture:

Mycobacterium phlei ATCC 19249, adapted to C C n-aIkane, was incoluatedfrom sloping agar cultures on to a culture medium 'of the followingcomposition:

Difco Bacto Nutrient Broth* 23 g.

C, C n-alkanes 10 ml. v a Trace element solution 1 ml.

Distilled water 1000 ml.

'Difco Manual, 9" edition.

Trace element solution: 11 g. ZnSO .7H O; 6 g. MnSO .4I-I O; l g. FeSO.7I-I O; 0.3 g. CoSO .7I-I O; 0.04 g. CuSO .5I-l O; 0.06 g. H BO 0.01 g.K1; 5 g.

33,20B-dihydroxypregn--ene Nrnno 2 g.

KH PO 1 g.

MgSO .7 H O 0.2 g.

KCl 0.2 g.

EeSO .7 H O 0.001 g. Tap Water, pH 7.0 1000 ml.

The cultures were incubated for days at 28C. with aeration and stirring.From the thus-obtained cultures, there were taken plate-cultures on agarplates which contained only 3B,20B-dihydroxypregn-S-ene as the carbonsource. The plate-cultures were repeated several times, by which meansthe steroid content in the agar plates was increased. Finally, furtherplatecultures were used in which the agar contained onlyB-hydroxy-9-oxo-l ,2,3,4, 1 0,l9-hexanor-5,9-secopregnan-S-oic acid'asthe carbon source. Those cultures which showed no growth on these plateswere used for the fermentation. In order to store the cultures, asuspension in sterile 10 percent skim milk was prepared from the biomassand was filled into 2 ml. ampoules and stored under liquid nitrogen.

Example 2 in tap water. pH 7.0 (adjusted with sodium hydroxide), wasinoculated with the contents of 2 ampoules prepared according toExample 1. The culture was incubated for 48 hours at 28C. with shaking.This preculture .was used to inoculate 8 liters of nutrient medium in asmall fermenter following which incubation was carried out for 2 days at28C. with stirring and aeration (640 r.p.m., 2 liters air/minute), thepH value being adjusted to pH 7.0 by the addition of 28 percent sodiumhydroxide. The contents of this fermenter were used as the inoculant for180 liter fermenter containing the same nutrient solution. After theinoculation, incubation was carried out at 32C., 1,100 r.p.m. and anaeration rate of l00-130 liters/minute. After 27 hours, the glucosecontent of the nutrient medium had fallen to 0.2 percent by weight. Atthis point, 180 g. of 3 {3,2OB-dihydroxypregn-S-ene dissolved in 3600ml. of dimethyl sulfoxide were added. The reaction of the steroid wasfollowed by thin layer chromatography carried out on samples. The pH washeld at between 6.5 and 7.0 by the addition of sodium hydroxide.

After 63 hours, the steroid used as the starting material had completelyreacted, whereupon another 180 g. of 3 B,20B-dihydroxypregn-S-ene indimethyl sulfoxide were added to the fermentation solution. Aftercomplete reaction of the steroid hours), a third addition of 180 g. ofsteroid was carried out. After 155 hours, all of the steroid hadreacted.

The fermentation mass was centrifuged, rinsed with 20 liters of waterand adjusted to pH 2.0 with concentrated aqueous hydrochloric acid. Thesolution was extracted twice with the same volume and once with half thevolume of methylene chloride. The extracts were concentrated in a rotaryevaporator and worked up by countercurrent extractionv The yield of20,8-hydroxy-9- oxo-1,2,3,4, l0,l9-hexanor-5,9-seco-pregnan-5-oic acidamounted to 82 percent. 10 percent of9,20-dioxo-1,2,3,4,l0,l9-hexanor-5-seco-pregnan-5-oic acid was also isolated.

EXAMPLE 3 20B-Hydroxy-9-oxo-l ,2,3,4,10,l9-hexanor-5,9-secopregnan-S-oic acid is converted to 20beta-hydroxydeA-pregn-9-en-5-one as follows:

A solution of 13.4 g of 20beta-hydroxy-9-oxo-1,2,3,4,1O,19-hexan0r-5,9-seco-pregnan-5oic acid and 4 g of sodiumacetate in 250 ml of acetic anhydride is boiled at reflux under nitrogenfor 4 hours. The acetic anhydride is removed in vacuum, the residuetwice evaporated with toluene and finally taken up in 750 ml ofmethylene chloride. The methylene chloride solution is washed five timeswith water, dried over Na SO and evaporated in vacuum. The resulting 7-(1R-acetoxyethyl)-6a-methyl-3(2H)-oxol,6,6a,7,8,9,9a,9b-octahydro-cyclopenta[f]l benzopyran is directly employed in the next step.

A solution of 8.8 g of 7-(lR-acetoxyethyU-bamethyl-3(2H)-oxo-1,6,6a,7,8,9,9a,9b-octahydrocyclopenta[f][llbenzopyranin 250 ml of absolute ether is slowly treated with 60 ml of a 0.5 Nethereal solution of ethyl magnesium bromide at 1 5. After 24 hours, thereaction mixture is poured onto ice-cold saturated ammonium chloridesolution. The ethereal solution is washed with water (1 X), ice-cold 0.5N caustic potash (2 X) and again with water (2 X). The organic phase isdried over sodium sulfate, the solvent is removed in vacuum and theresidue is filtered through a column of 25 g of silica gel.

1.1 g of the thus obtained crude product are stirred with a solution of3 g of potassium hydroxide in ml of methanol and 10 ml of water for 15hours at room temperature. The reaction mixture is poured onto icewaterand extracted with ether. The ethereal extract is washed neutral withwater, dried and evaporated. Chromatography of the residue on silica gelyields pure 20beta-hydroxy-deA-pregn-J-en-5-one.

We claim:

l. A process for the manufacture of 20betahydroxy- 9-oxo-l,2,3,4,10,19-hexanor-5.9-seco-pregnan-5-oic acid of the formula:

7 2. A process according to claim 1, wherein used spontaneous mutantswhich show no growth on a nutrient medium containingbeta-hydroxy-9-oxol,2,3,4,10,19-hexanor-5,9-seco-pregnan-5-oic acid asthe carbon source.

3. The process of claim 1 wherein the fermentation is carried outaerobically. I

4. The process of claim 1 wherein the fermentation I is carried out at apH of from 6 to 7.

1. A PROCESS FOR THE MANUFACTURE OF20BETA-HYDROXY-9-OXO1,2,3,4,10,19-HEXANOR-5,9-SECO-PREGNAN-5-OIC ACID OFTHE FORMULA:
 2. A process according to claim 1, wherein there are usedspontaneous mutants which show no growth on a nutrient medium containing20beta-hydroxy-9-oxo-1,2,3,4,10,19-hexanor-5,9-seco-pregnan-5-oic acidas the carbon source.
 3. The process of claim 1 wherein the fermentationis carried out aerobically.
 4. The process of claim 1 wherein thefermentation is carried out at a pH of from 6 to 7.